Biosensors and Bioelectronics 24 (2009) 1292–1297

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Homogenous growth of gold nanocrystals for quantification of PSA protein biomarker Cuong Cao a , Xinxing Li a , Jeewon Lee b , Sang Jun Sim a,∗ a b

Nano-optics and Biomolecular Engineering National Laboratory, Department of Chemical Engineering, Sungkyunkwan University, Suwon 440-746, Republic of Korea Department of Chemical and biological Engineering Korea University, Seoul 136-713, Korea

a r t i c l e

i n f o

Article history: Received 1 June 2008 Received in revised form 21 July 2008 Accepted 22 July 2008 Available online 3 August 2008 Keywords: Gold nanoparticles Homogenous nanogrowth Protein biomarker Magnetic separation Solution phase

a b s t r a c t We report on another alternative sensing platform for the detection of protein biomarker (PSA–ACT complex) based on homogenous growth of Au nanocrystals in solution phase. The immuno-recognition event is translated into the gold nanoparticle growth signal which can be intuitively recognized by an unaided eye, or quantitatively measured by an UV–vis spectrophotometric analysis. Surface plasmonic signature and kinetics of the Au nanogrowth in the homogenous phase containing of HAuCl4 , AA, and CTAB have also been studied to provide suitable parameters for the immunoassay. As a result, detection limit of PSA–ACT complex was determined to be 10 fM. The result indicated that this is a very sensitive, robust, simple, and economic strategy to detect protein biomarkers, and it has great potential to detect other biological interactions. © 2008 Elsevier B.V. All rights reserved.

1. Introduction Detection of specific protein biomarkers for diagnosis and monitoring of cancer diseases is an extremely urgent issue. To accomplish this task, many studies have been devoted to the development of various signal transduction methods based upon optics (Shankaran and Miura, 2007), radioactivity (Lindstedt et al., 1990; Cuny et al., 1996), fluorescence (Soukka et al., 2003), electrochemistry (Fernandez-Sanchez et al., 2004), quartz crystal micro balance (Henne et al., 2006), piezoelectric cantilever (Lee et al., 2005), colorimetry (Liang et al., 2004; Nam et al., 2005), scanometry (Nam et al., 2003; Stoeva et al., 2006), or Raman spectroscopy (Cao et al., 2003). However, the development of simple and highly sensitive tools that enable real-time detection of biomarker species is still a significant driving force in biosensor research. Recently, gold nanoparticles (AuNPs) have been extensively utilized in fabrication of various biosensing systems owing to their excellent biocompatibility and their special characteristics. By coupling AuNPs to biomolecules such as antigens or antibodies, detection and quantification of the biological interactions have been implemented by advantages of AuNPs as optical labels (Ambrosi et al., 2007), electrochemical markers (Das et al., 2006; Zhou et al., 2006), or surface plasmonic amplifiers (Cao and Sim, 2007a,b). The

∗ Corresponding author. Tel.: +82 31 290 7341; fax: +82 31 290 7272. E-mail address: [email protected] (S.J. Sim). 0956-5663/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2008.07.046

use of Au nanoparticles as catalytic probes for the growth of the particles is another beneficial feature for the biosensing purpose, where the bio-recognition event is translated into AuNP growth signal (Moeremans et al., 1984; Lackie, 1996). Ordinarily, the sensing signal is strongly amplified by a nanogold probe coupled with silver enhancement where the reduction and deposition of silver ions are catalyzed by the gold nanoparticles (Henglein, 1989). The so-called silver staining method has provided numerous chip-based biosensing platforms with suitable readouts including those relying on light scattering (Sun et al., 2007; Xu et al., 2007), scanometry (Taton et al., 2000), colorimetry (Liang et al., 2004), and conductivity (Park et al., 2002). Although the silver enhancement method allows one to directly observe the reaction signals on a glass chip or on a nitrocellulose strip by the naked eye (Taton et al., 2000), the detection sensitivity is much lower than those performed by fluorescent, radioactive, and colorimetric assays. Other alternatives have been reported by several groups in which a mixture of HAuCl4 and reductants was utilized to enlarge the immune AuNPs immobilized on a nitrocellulose strip, and those methods offered better sensitivity for detection of immunoglobulin G (IgG) in comparison with the silver enhancement method (Ma and Sui, 2002; Su, 2005). Their data indicated that the sensitivity of IgG detection based on the growth of AuNPs was significantly increased to 10 pg/ml; this level already approached that of standard fluorescent or colorimetric techniques for immunoassays (Ma and Sui, 2002). Growth of aptamerfunctionalized AuNPs for optical detection of thrombin on a solid substrate is another illustration representing an amplification path

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by using a mixture of HAuCl4 , NADH and CTAB (Pavlov et al., 2004). Although the chip-based nanogrowth formats have been the focus of many studies for visualization and detection of biorecognitions, very few and preliminary have been dedicated to the evaluation of the merits of the catalytic Au nanoprobes for homogenous detection designs in solution. Xu et al. (2007) has developed a technique, which based on light scattering characteristics of silveramplified gold nanoparticles probes, to detect nucleic acids. Their results showed that the homogenous detection platform offered a simple, rapid, and sensitive assay with a limit of detection as low as fM level. However, it has not been used for monitoring protein–protein interactions, especially for those relying on the homogenous growth of Au nanoparticles by HAuCl4 (not the Au probe-silver enhancement). Hereafter, we wish to introduce another homogenous detection assay that incorporate the Au nanocrystalline growth with the use of magnetic microbeads (MMPs) to quantify a target protein biomarker based on sandwich immune reactions. To examine the assay, prostate specific antigen (PSA–ACT complex) has been chosen as the typical target analyte because of its valuable signature in the diagnosis of prostate cancer (Lindstedt et al., 1990; Cuny et al., 1996). A sandwich format of AuNP/PSA–ACT/MMP was fabricated; after the immunoreactions, the target analytes were collected and separated by taking advantage of MMPs while the growth of AuNPs plays a role of the color developer. Therefore, the quantitative information of target analyte is translated into a colorimetric signal which can be intuitively recognized by an unaided eye, or quantitatively measured by a UV–vis spectrophotometric analysis. The result shows that this is a very sensitive, robust, and versatile strategy to detect PSA antigens, the lowest detecting concentration was acknowledged to be about 10 fM (1 pg/ml) with high specificity. The highly sensitive method vanquishes all detection limits of commercially available tests for PSA so far, and also can be expanded to detect other protein biomarkers at low concentrations.

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stirring vigorously. Upon the addition of NaBH4 , the solution color turned into reddish orange, and it was continued being stirred for the next 1 min. Next, the resulting mixture was aged for 2–4 h in order to allow the unreacted NaBH4 to be completely hydrolyzed. This method yields spherical Au nanoparticles with an average diameter of about 5–6 nm as measured by transmission electron microscopy (TEM). 2.3. Preparation of Au immunoprobes The Au immunoprobes, which manifest gold-labeled PSA mAb, were prepared by physisorption following a method reported previously with some modifications (Ambrosi et al., 2007). Au nanoseed solution was adjusted to pH 9 with 0.1 M K2 CO3 solution. Thereupon, adsorption isotherms were constructed for PSA mAb to determine the minimum amount of protein that was necessary to coat the exterior of the Au nanoseeds. 400 ␮l of colloidal Au nanoseeds was mixed with a 50 ␮l series of PSA mAb solutions of increasing concentration (up to 1 mg/ml), and the mixture was incubated at room temperature for 20 min. Then, 400 ␮l of 10% NaCl was added and rapidly mixed. If the protein was not adsorbed and the gold nanoseeds were not stabilized, aggregation of the gold granules was indicated by a color change from red to light blue. The minimum amount of protein that would prevent this color change was used. Afterwards, the PSA mAb-Au immunoprobes was synthesized by the addition of the minimum amount of PSA mAb plus 10% to 10 ml of pH-adjusted Au nanoseeds followed by gentle mixing for 30 min. Then, 0.5 ml of freshly made and prefiltered (0.45 ␮m Millipore) containing 10% BSA and 1% Tween-20 were added to prevent aggregation of the PSA mAb-Au immunoprobes and to block the non-specific adsorption of other proteins. The protein-labeled gold probes were centrifuged at 15,000 × g for 1 h at 4 ◦ C to remove the excess of antibodies. The clear to pink supernatant was gently discarded, and the labeled gold pellet was resuspended in 10 ml of 0.01 M PBS buffer (pH 7.4). The Au immunoprobe solution was stored at 4 ◦ C for up to 1 week without loss of activity.

2. Materials and methods 2.4. Preparation of MMP immunoprobes 2.1. Chemicals and materials Epoxy-coated magnetic microbeads (M-270 Epoxy) were purchased from Dynal Biotech (Invitrogen, USA). Human prostatic specific antigen (PSA–ACT complex), PSA–ACT complex monoclonal antibody (PSA mAb), and goat PSA polyclonal antibody (PSA pAb) were supplied by BiosPacific, Inc. (Emeryville, CA, USA). Bovine serum albumin (BSA), human immunoglobulin G (IgG), fibrinogen from human plasma, hydrogen tetrachloroaurate (III) trihydrate (HAuCl4 ·3H2 O, 99.9%), trisodium citrate, sodium borohydride (NaBH4 ), cetyltrimethylammonium bromide (CTAB), ascorbic acid (AA), phosphate buffer saline pH 7.4 with Tween-20 (PBS buffer) were obtained from Sigma–Aldrich. Other essential inorganic reagents were supplied by Pierce, Sigma, Aldrich, or Fluka unless otherwise stated. All chemicals were used as received, and all of the chemical solutions were prepared in ultra pure water (18.2 m cm) when needed.

The experimental steps for the preparation of MMP immunoprobes were carried out by coating PSA pAb with Dynabeads M-270 Epoxy according to the manufacturer’s protocol with some modification. In brief, after washing and equilibration of the MMPs, 0.1 M sodium phosphate buffer (pH 7.4) was added to obtain a concentration of 15 mg/ml (approximately 109 beads per ml). Then, 400 ␮l of 1 mg/ml PSA pAb solution was mixed with 1 ml of the bead suspension (15 mg/ml). To enhance binding, a final concentration of ammonium sulfate of 1 M was supplemented to the solution. The mixture was gently mixed and incubated for about 48 h at 4 ◦ C to ensure covalent coupling. After incubation, the MMP immunoprobes were blocked with 0.2% BSA solution, separated by a 3000G permanent magnet, and washed with PBS buffer containing Tween20 for three times. 2.5. Preparation of growth solution for the growth of Au nanocrystals

2.2. Preparation of Au nanoseeds Gold nanoseeds were prepared by reducing HAuCl4 with NaBH4 and stabilized with sodium citrate according to a procedure reported previously (Millstone et al., 2005). Briefly, 0.5 ml of 0.1 M NaBH4 was added into a solution containing 0.5 ml of 10 mM HAuCl4 , 1 ml of 5 mM trisodium citrate, and 17.5 ml DI H2 O while

10 ml solution consisting of final effective concentrations of 0.25 mM HAuCl4 and 0.1 M CTAB was prepared in a 20 ml vial to yield a clear orange color. The solution was gently mixed by inversion of the vial after the addition of every component. When applying the gold nanocatalytic growth, 10 mM AA solution was added into the solution and stirred just before use.

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2.6. Quantification of PSA–ACT complex by the homogenous growth of Au nanocrystals In a typical experiment, a sandwiched immunoassay of Au immunoprobe/PSA–ACT/MMP immunoprobe was fabricated, and then collection of the Au immunoprobes was performed by a magnetic separation and followed by introduction of the growth solution for the Au nanogrowth. Briefly, 100 ␮l of 1 mg/ml MMP immunoprobes was incubated with 300 ␮l of various concentrations of PSA–ACT complex ranging from 0 pg/ml to 100 ng/ml in an eppendorf tube. The reaction was kept for 1.5 h at 37 ◦ C with gentle mixing. Following the washing step of these beads with 0.01 M PBS buffer (pH 7.4) three times, a 200 ␮l volume of Au immunoprobes was added and incubated for the next 45 min at 37 ◦ C. When the immunoassay finished, the Au immunoprobe/PSA–ACT/MMP immunoprobe complexes were separated by using the permanent magnet and washed with the same buffer to remove the excess of Au probes. After collection of the pellet, the Au probes were dissociated from the complexes by using 60 ␮l of 50 mM NaCl/1 M NaOH elution solution. Finally, the upper aqueous solution containing the Au probes was obtained and transferred to a 1 ml cuvette following addition of 1ml of growth solution. It should be noted that addition of the mixed solution of 50 mM NaCl/1 M NaOH will change pH of the growth solution from 2.6 to 11.8; however, this does not strongly influence the stabilization of the growing particles. The growth of Au nanocrystals and solution color developed by catalysis of the Au probes were recorded by a high resolution transmission electron microscopy (HR-TEM, JEOL JEM-3011 operated at 300 kV) and an UV–vis scanning spectrophotometer (DU 730, Beckman Coulter operated at resolution of 1 nm), respectively. 3. Results and discussion The principle of the quantitative assay of protein biomarker is illustrated in Fig. 1. The detection involves the sandwiching

of the target protein between the Au immunoprobes and MMP immunoprobes (step 1). Following the separation of the sandwiched immunocomplex by a permanent bar magnet (step 2), a mixed solution of 50 mM NaCl/1 M NaOH is added to elute the immune reactions (step 3). Subsequently, the Au immunoprobes are separated from the MMP immunoprobes by the magnet (step 4), and then transferred to a cuvette where the Au growth is performed by adding a volume of growth solution (step 5). In this stage, the Au probes act as self-catalysts by receiving and transferring the electrons (produced from reduction of AA) to Au ions; and catalyzing for the deposition of Au ions on the particle surface for their crystalline growth. The enlarging process will be catalyzed continuously by the Au probes until the Au ions and the hydrated electrons are exhausted. This will lead to changes in optical properties of the AuNPs corresponding to their growth that will be easily recorded by a UV–vis spectrophotometer, or even by the naked eye. The deposition and crystallization processes will depend on the amount of Au probe, or in other words, it will depend on the concentration of target protein molecules providing a quantitatively monitoring method for protein biomarkers. For the homogenous growth of Au nanocrystals, CTAB and AA have been widely utilized in a seeding method due to the fact that AA can reduce CTAB–Au3+ complex to Au+ which could be subsequently reduced to Au◦ at the surface of small Au seeds (Johnson et al., 2002; Sau and Murphy, 2004), resulting in the size growth of the AuNPs as well as the enhancement of optical absorbance AA

Au3+ + Au0n=1 (seed, atom = 1) + 3e− −→Au0n=2 In this study, the Au probes act as catalysts and nuclei for the growth of particles while CTAB plays a role of the stabilizing surfactant. Because it is well known that metallic nanoparticles are unstable due to high surface energy, they have a tendency to aggregate leading to an alteration of optophysical properties of the nanostructures. Therefore, the use of stabilizer is very significant

Fig. 1. Schematic representing general procedure of the Au nanogrowth-based immunoassay for ultrasensitive quantification of protein biomarker consisting of five steps thoroughly described in the text. (For interpretation of the references to color in the artwork, the reader is referred to the web version of the article.)

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to prevent the aggregation and to maintain the stability of AuNPs in aqueous solution, especially for assays that are based on the unique characteristics (light scattering, light absorption, etc.). 3.1. Growth of Au immunoprobes in aqueous solution Obviously, the optical properties of metal nanoparticles are strongly influenced by their size and shape (Kelly et al., 2003). By varying concentration of reagents, many sizes and shapes of AuNPs have been obtained by using the seed-mediated method, and their surface plasmon absorbance patterns have also been acknowledged with some variances (Sau and Murphy, 2004). As far as we know, the homogenous growth using 10 mM AA and 0.1 M CTAB for deposition Au ions on the surface of the protein-coated Au probes was performed for the first time in aqueous solution phase; therefore it was necessary to address the surface plasmonic signature which is the read-out base of this colorimetric detection. Fig. 2 shows UV–vis scanning spectra illustrating the growth of Au immunoprobes in aqueous solution. After coating the exterior with PSA mAb, 100 ␮l of Au immunoprobes was added to a cuvette containing 1 ml of the growth solution, and the UV–vis spectra were scanned after every 2 min interval. As clearly seen, the particle growth happens almost instantaneously after 2 min indicated by an increasing in intensity of the maximum absorbance values at 529 nm. During the particle growth, the plasmon absorbance spectra are slightly shifted to the longer wavelengths with sharper peaks showing that the Au nanocrystals are enlarging in size as well as the stabilization of forming AuNPs in solution. The stability of the newly produced AuNPs greatly influences the consistency of the final results because of the different aggregation states of AuNPs which can results in distinctive color changes (Ambrosi et al., 2007). Since the typical maximum absorption peaks (max ) are fixed and stably centered at around 565 nm after 18 min of the catalytic growth process (see the inset), thus the maximum absorbance at wavelength of 565 nm is relatively chosen as an analytical parameter for quantitative justification of the particle growth throughout the assay. Morphology of the Au immunoprobes before and after applying the growth solution was also investigated by TEM studies to prove that the particles were actually enlarged in size. As shown in Fig. 3A, the Au immunoprobes have an average size of about 5–6 nm. After the treatment of growth solution containing 0.25 mM HAuCl4 , 10 mM AA, and 0.1 M CTAB, the Au immunoprobes were fully grown to a dominant size of about 35 nm. Owing to the stabilizing benefit

Fig. 2. UV–vis scanning spectra illustrating the growth of Au (5–6 nm) immunoprobes in aqueous solution. The spectra were measured after every 2 min interval, and their intensities increased with the time course. Maximum absorbance peaks are slightly shifted from 529 nm and stably reached to 565 nm after 18 min of growth (inset). (For interpretation of the references to color in the artwork, the reader is referred to the web version of the article.)

Fig. 3. TEM images of the Au immunoprobes (A) and (B) the AuNPs enlarged by 0.25 mM HAuCl4 , 10 mM AA, and 0.1 M CTAB in aqueous solution.

of CTAB, the enlarged AuNPs were well-dispersed and very stable in solution for several weeks without any color loss although their shape was not very homogenous (Fig. 3B). In order to exploit the quantitative capabilities of this system, it is essential to investigate kinetic behaviors of the catalytic growth to comprehend how the time affects the colorimetric analysis under the controlled contexts. Various initial concentrations of Au immunoprobes were added to a 1 ml volume of the growth solution and experimented under the same conditions. Fig. 4A shows that the color of solution is gradually changed from colorless to violet with the time course, and the color intensities can be discriminated by the unaided eye. However, it is clearly seen that the color tone will be easier to differentiate if the time of growth is longer. In correlation, the kinetics of particle growth is shown in Fig. 4B where absorbance of the gold plasmon band at 565 nm was used to monitor the growth as a function of time. As indicated by UV–vis spectroscopy, the particle growth initiated as soon as the addition of growth solution followed by the saturation phase. The maximum absorbance values at 565 nm were obtained after 15 min for the 5.2 × 10−7 M solution of Au immunoprobes, and extended up to 50 min as the concentrations of Au immunoprobes decreased, e.g. the 0.65 × 10−7 M sample. The rate of particle growth is highly dependent on and proportional to the initial concentrations of Au immunoprobes proving that quantitatively monitoring method of protein biomarkers based on the amount of Au probes is highly feasible. In a practical detection assay, it is important to identify an essential time growth that is proportional to the target analyte concentration. Therefore, owing to the fact that the produced particles will not aggregate, and their absorbance signals will be proportional to the number of initial Au probes after the saturation

Fig. 4. (A) Color images of solution growth containing different concentrations of Au immunoprobes: 1 = 0.65 × 10−7 M; 2 = 1.3 × 10−7 M; 3 = 5.2 × 10−7 M. The color is gradually changed with the time and proportional to the concentrations of Au immunoprobes. (B) Kinetic plots of gold plasmon absorbance at 565 nm versus time. (For interpretation of the references to color in the artwork, the reader is referred to the web version of the article.)

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Fig. 5. Logarithmic correlation corresponding to the Au nanogrowth immunoassay for the detection of PSA–ACT complex.

phase, a growing time of 50 min was selected to obtain the highest absorbance intensities from each sample. 3.2. Detection of PSA protein biomarker mediated by the growth of Au nanocrystals in aqueous solution After learning parameters about the surface plasmonic signature and kinetics of Au growth in the homogenous phase, the assay was expanded to a practical detection of protein biomarker. PSA–ACT complex with various concentrations spanning from 0 pg/ml to 100 ng/ml was investigated. The low range of analyte concentration was chosen for these reasons. Firstly, detection of protein biomarkers with higher concentration range can be easily approached by many simple and widely used methods such as ELISA (Lindstedt et al., 1990; Cuny et al., 1996) or SPR (Cao et al., 2006; Cao and Sim, 2007a,b). Secondly, superior sensitive assays are highly desired due to their significant contributions to early diagnosis of cancerous diseases. Following the incubation steps with Au immunoprobes and MMP immunoprobes, the immune reactions were dissociated by using an elution solution, and the Au probes were separated and collected by means of magnetic force. After 50 min of complete growth, color of solution was developed and measured by an UV–vis spectrophotometer. Fig. 5 shows the logarithmic correlation between relative absorbance values and the PSA–ACT complex concentrations, where the signals were deducted from the blank measurements. Although it could not be detected at concentrations lower than 1 pg/ml, the detection of PSA–ACT complex was possible in the concentration range of 1 pg/ml to 100 ng/ml in which the maximum absorbance signals were linearly proportional to the PSA–ACT complex concentration. The plot indicates that the PSA–ACT complex concentrations were determined by the homogenous growth of Au nanocrystals over a wide range. Due to the subtraction of the blank signal from the PSA-containing measurements, the limit of detection (L.O.D.) was therefore supposed to be the smallest concentration, which could be completely distinguished from the background, as low as 1 pg/ml (about 10 fM). 3.3. Investigation of non-specific binding of different proteins in the detection assay In any immunoassay for detection of protein, interaction between non-target proteins and the sensing materials could be a source of noise leading to aberrance of the final results. To investigate non-specific binding between the surface materials, BSA, IgG and fibrinogen were used. These are the most common proteins present in human serum at high concentration. As shown in

Fig. 6. Non-specific binding of different proteins in the detection assay. The adsorptions of BSA, IgG, and fibrinogen were negligible. The solid line represents detection limit of PSA–ACT complex. (For interpretation of the references to color in the artwork, the reader is referred to the web version of the article.)

Fig. 6, a series of control experiments with increasing concentrations were studied, and the non-specific adsorptions of BSA, IgG and fibrinogen were not substantial. When the concentrations of these foreign proteins were increased from 1 to 100 ng/ml, the corresponding absorbance values fluctuated and increased nonsignificantly which were still very far below the values of PSA–ACT samples at the same concentrations, or even lower than the L.O.D. of PSA detection marked as the solid line. The low nonspecific binding of these proteins could be explained due to the sensing immunoprobes which were effectively blocked during preparation, and the fact that the hydrophilic nature of the immobilized antibody layers has the ability to render surface biocompatibility of the Au and MMP immunoprobes. Several methods have been implemented to achieve better sensitivity of PSA detection (Cao et al., 2006; Cao and Sim, 2007a,b). This study achieved the highest sensitivity over a wide range of detection that has not been obtained before by using SPR-based biosensors. Although the femtomolar quantification is not sensitive as compared with those obtained from the biobarcode assay (Nam et al., 2003; Stoeva et al., 2006), its simplicity and applicability do have considerable contributions in analysis of bio-recognition or clinical diagnostics. Indeed, this solution-based assay harnessing Au immunoprobes offers several surpassing advantages over the current chip-based formats. Firstly, higher accessibility of target analytes to Au immunoprobes can be obtained due to the fact that the Au probes have a high surface to volume ratio in solution as well as the homogeneousness of the mixing condition. Secondly, it provides a possibility to exploit useful benefits of MMPs in a sandwich format where the MMPs can be easily directed by an external magnetic force; and therefore, separation, purification, and enrichment of target proteins in a reaction mixture are approachable when needed (Kouassi et al., 2007). Thirdly, the homogeneity of the colorimetric solution assay offers higher suitability towards commercialized cuvette-based spectrometers where the absorbance/transmission signals are supposed to be more identical than those measured on heterogeneous chip-based formats. Finally, potentials of real-time monitoring, automation, and risk reducing from contamination make the homogenous assay a fascinating, versatile detection of biological recognitions where kinetic modes and aseptic conditions are highly desired. 4. Conclusions A novel detection of protein biomarker using homogenous growth of Au nanoprobes incorporated with beneficial uses of MMPs has been described. The catalytic growth of Au nanoprobes

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was performed with the mixed solution of 0.25 mM HAuCl4 , 10 mM AA, and 0.1 M CTAB in homogenous solution phase. This assay was very simple and straightforward to perform; as a result, the sensitivity was obtained as low as 10 fM for the detection of the famous PSA protein biomarker. Although simple in design and concept, the strategy introduced is highly sensitive, specific, and highly applicable to analysis of other protein biomarkers as well as a wide range of fields. Moreover, it still offers suitability to detect target analytes with an unaided eye or with the use of an inexpensive readout device. Therefore, the method would open up possibilities for future use of homogenous Au growth as biosensing platform to detect various cancer-associated proteins qualitatively or quantitatively. Expansion of the assay to detect other protein biomarkers is in progress. Acknowledgment This work was supported by the Korea Science and Engineering Foundation (KOSEF) National Research Laboratory (NRL) Program grant funded by the Korea government (MEST) (grant no. R0A2008-000-20078-0/ROA-2007-000-20084-0) of the Republic of Korea. References Ambrosi, A., Castaneda, M.T., Killard, A.J., Smyth, M.R., Alegret, S., Merkoci, A., 2007. Anal. Chem. 79, 5232–5240. Cao, C., Sim, S.J., 2007a. Biosens. Bioelectron. 22, 1874–1880. Cao, C., Sim, S.J., 2007b. J. Microbiol. Biotechnol. 17, 1031–1035. Cao, Y.C., Jin, R., Nam, J.M., Thaxton, C.S., Mirkin, C.A., 2003. J. Am. Chem. Soc. 125, 14676–14677. Cao, C., Kim, J.P., Kim, B.W., Chae, H., Yoon, H.C., Yang, S.S., Sim, S.J., 2006. Biosens. Bioelectron. 21, 2106–2113. Cuny, C., Pham, L., Kramp, W., Sharp, T., Soriano, T.F., 1996. Clin. Chem. 42, 1243–1249.

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